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Histoplasma capsulatum, a thermally dimorphic pathogenic fungus, is the etiologic agent of histoplasmosis (1, 5). H. capsulatum is a cosmopolitan species, but in Asian countries such as Indonesia, Thailand, India, and Myanmar, the fungus is believed to have a limited regional distribution (1). Our preliminary survey on fungal infections in Thailand indicated that the recent increase in AIDS patients has led to an increase in infections caused by this pathogenic fungus. To date, however, there have been no reports on genomic studies of H. capsulatum strains in Thailand.

Since diverse DNA fingerprinting patterns of H. capsulatum isolated from clinical specimens or soils in various regions in America have been reported (6, 10), we were interested in the DNA genomic patterns of clinical isolates of this fungus in Thailand. From 1993 to 1997, we isolated 13 strains of H. capsulatum from clinical specimens, including those from AIDS patients, in Thailand. In this study, the results of genomic analysis by PCR-based random amplified polymorphic DNA (RAPD) analysis compared with those for a reference North American strain (H. capsulatum IFM 46159 [original strain no., G217-B]) (3) are reported.

The history of 13 strains isolated in Thailand is shown in Table 1. Each strain was identified by its dimorphic nature, which includes mold-like growth on brain heart infusion (BHI; Difco) agar at 27°C and oval or elongated yeast cells on BHI agar medium at 37°C. Final species confirmation was done by the direct microscopic observation of special tuberculate macroconidia on BHI medium at 27°C (1, 5).

Chromosomal DNA of 14 strains was extracted by a modified method of Zhu et al. (12) and Mekha et al. (7). The strains were grown on BHI agar at 37°C for 1 week. Three or four loopfuls of yeast cells from the agar slants were suspended in 500 µl of TE buffer (100 mM Tris-HCl [pH 8.0], 1 mM EDTA) in an Eppendorf tube (1.5 ml) and kept in a heater at 80°C for 30 min to kill the fungus. After it was shaken, the suspension was centrifuged at 12,000 rpm and the sediment was mixed with 0.5 ml of extraction buffer (100 mM Tris-HCl [pH 9.0] with 40 mM EDTA), 0.1 ml of 10% sodium dodecyl sulfate, and 0.3 ml of benzyl chloride. After they were shaken with a vortex mixer, the fungal cells were incubated at 60°C for 30 min and then broken by a beadbeater (Wakenyaku Co., Kyoto, Japan) for 20 s. After centrifugation at 11,000 × g at 4°C for 5 min, the supernatant was transferred into a new Eppendorf tube, 3 M sodium acetate was added with 1/10 volume of the aqueous solution, and the mixture was cooled at 0°C for 10 min. DNA was precipitated with 0.5 ml of 2-propanol (70°C, 3 h), and then the precipitate was washed with 70% ethanol, dried, and resuspended in 100 µl of TE buffer. The DNA concentration was determined by UV monitor (Bio-mini; Atto, Tokyo, Japan).

For the RAPD analysis, three primers were used: R-1, 5'-A TGGATCGGC-3'; R-2, 5'-ATTGCGTCCA-3'; and R-3, 5'-TCACGATGCA-3'. These were prepared based on the reports of Goodwin and Annis (4) or on information from our preliminary experiments. A specially purified RAPD PCR primer for DNA sequencing, SPP-seq, 5'-ATGGATC(GC)(GC)C-3', was used because this primer produced more PCR bands than any primer tested in our preliminary experiments. Amplification reactions were carried out in a volume of 30 µl of distilled water containing 2.5 µl of primer (20 pM), 2.5 µl of genomic DNA (1 µg/ml), and one PCR bead (Ready-to-GO PCR bead; Pharmacia Biotech). The PCR was performed by initially heating the samples at 94°C for 4 min; this was followed by 35 cycles of denaturation at 94°C for 2 min, annealing at 32°C for 2 min, extension at 72°C for 2 min, and the final extension at 72°C for 10 min with a thermoreactor.

The normal PCR with the newly prepared primer set was performed by initially heating the samples at 94°C for 10 min; this was followed by 25 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min, and a final 10 min extension step at 72°C. All reaction products were characterized by electrophoresis on 1.5% agarose gels in 1× TBE buffer at 80 V for 90 min and then stained in 0.5 µg of ethidium bromide solution per ml.

Thirteen strains of H. capsulatum isolated in Thailand, mainly from AIDS patients, had characteristic DNA fingerprinting patterns for each primer as shown in Fig. 1. These patterns of Thai isolates were very similar to each other in all primers, except for one strain (NIH 36-502-23). Although the differences in the DNA fingerprinting patterns were minor, Thai isolates could be classified into two to three groups for each primer.

View larger version (90K): [in this window] [in a new window]   View larger version (89K): [in this window] [in a new window]   View larger version (102K): [in this window] [in a new window]   FIG. 1.   RAPD fingerprint band patterns of 13 strains of H. capsulatum isolated in Thailand with primer R-1 (a), primer R-2 (b), and primer R-3 (c). DNA ladder marker was used as the molecular size standard. H. capsulatum IFM 46159 (G217-B) was used as a reference strain.

With primer R-1, Thai isolates were classified into four groups (designated A to D) on the basis of the minor differences of the PCR bands as shown in Fig. 1a. A graphic representation of the four DNA fingerprint patterns is shown in Fig. 2. While patterns A, B, and C were similar, pattern D (strain NIH 36-502-23) was somewhat different. Isolates exhibiting patterns A and B appeared to be more prevalent types of clinical specimens in Thailand. The reference strain had a DNA fingerprinting pattern that was similar to those of Thai isolates with this primer, but some bands which commonly exist in Thai isolates were missing from the reference strain, indicating a clear difference (Fig. 1).

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Graphic representation of the four DNA fingerprint patterns obtained from clinical isolates of H. capsulatum in Thailand with primer R-1. Some bands common to all strains were omitted.

With primer R-2, the DNA fingerprinting patterns of the Thai isolates were also very similar, but they could be divided into three types (types 1, 2, and 3) on the basis of minor differences (Fig. 1b). Most of the isolates exhibited pattern 1 with this primer. The DNA fingerprinting patterns of the reference strain for this primer were clearly different from those of the isolates.

With primer R-3, most of the Thai isolates also showed a similar DNA fingerprinting patterns (pattern a), but strain NIH 36-502-23 showed a different pattern (pattern b) (Fig. 1c). The reference strain has several PCR bands common to all Thai strains and one which clearly differentiates it from the Thai isolates with this primer.

Data from all three primer sets suggested that Thai isolates are generally genetically homogeneous, while strain NIH 36-502-23 is significantly different from them. The strain was isolated from bronchoalveolar lavage fluid (BAL) of an AIDS patient in Bamrasnaradura Hospital, Central Thailand (Table 1). Interestingly, although another strain, NIH 36-520-41, was isolated from a blood sample from the same patient (on different days), the DNA fingerprinting patterns of the two strains were different from each other. These data may suggest the possibility of mixed infection due to genetically different H. capsulatum strains in the patient.

The DNA fingerprinting pattern of the U.S. reference strain with all three primers clearly differed from those of all the Thai isolates tested, including NIH 36-502-23.

The reason for the genetic homogeneity of the Thai isolates of H. capsulatum compared with the American reference strain is not clear but may be their geographical separation.

Molecular techniques such as restriction fragment length polymorphisms and RAPD have provided powerful tools for the direct analysis of the genomes of many pathogens (6, 8, 9, 11). Spitzer et al. (10) divided clinical and soil isolates of H. capsulatum into four classes based on the restriction fragment length polymorphisms of mitochondria or ribosomal DNA. We confirmed here by RAPD that Thai strains of this fungus are genetically homogeneous. Further comparative studies with RAPD and other molecular techniques and many isolates of H. capsulatum are necessary to fully delineate the homogeneity of Thai strains and provide useful epidemiological information on histoplasmosis in Thailand.

Systemic histoplasmosis has been reported in patients with AIDS who did not reside in regions where histoplasmosis is endemic. These reports suggest that the infection is due to the reactivation of previously acquired H. capsulatum infection in these patients (6). In the present studies, it was found that the DNA fingerprinting patterns of Thai isolates are distinct from those of the North American isolate. Therefore, information on the genomic characteristics of Thai H. capsulatum strains may be useful for elucidating a reactivation hypothesis of histoplasmosis in AIDS patients (6), because opportunities for the relocation of this fungus to areas of nonendemicity and for the infection of individuals positive for human immunodeficiency virus with Asian types of this fungus will increase as people travel and barriers to trade are removed.

H. capsulatum grows very slowly in most media tested and requires 1 to 2 weeks to mature. Isolates may be pleomorphic, and the presence of a variety of morphological forms makes identification confusing (1, 10). In fact, in our laboratory, we sometimes found no typical tuberculate macroconidia. Another AIDS-associated fungus, Penicillium marneffei, is endemic to Thailand and produces morphological features (in tissues) that are indistinguishable from those of H. capsulatum (2, 7). Since H. capsulatum is a treatable disease, and delay in treatment can be fatal, early diagnosis and subsequent therapy are very important (1, 3, 5). Therefore, a PCR primer set which can be used for the rapid identification of H. capsulatum strains should be required in clinical laboratories for direct specimen testing, culture identification, or both. One PCR fragment was extracted which appears to exist in the DNA fingerprinting patterns of all H. capsulatum strains used in these studies (about a 700-bp band in the PCR products when SPP-seq RAPD primer was used), and sequencing was accomplished by subjecting a RAPD PCR product to phenol-chloroform extraction and precipitation with ethanol. The DNA fragment was then sequenced with a dye terminator reagent kit including Taq polymerase and the protocol recommended by the manufacturer (model 373 automated DNA sequencer; Applied Biosystems, Tokyo, Japan). The length of the DNA sequence was found to be 705 bp. A comparison of the determined nucleotide sequence with the known sequences in the GenBank database showed no previously reported high homologous sequences. We prepared the following PCR primer set (Hc-T-s, 5'-GAATCTCAGGTCAATCGGTG-3'; and Hc-T-a, 5'-AGTTTCGCTGGAGTCAATTC-3') based on the sequence information of the 705-bp fragment. This new PCR primer set was designed to amplify a 373-bp area of DNA in H. capsulatum genomes. When the genomic DNAs from humans, Aspergillus fumigatus Tsukuba, Candida albicans ATCC 90028, Cryptococcus neoformans NIH 18, and P. marneffei ATCC 201698 were tested with this PCR primer set, amplification of the 373-bp fragment was confirmed only in H. capsulatum genomes, and no PCR products from other fungi or human genomes tested were observed (Fig. 3). The primer set was also 100% successful in tests of the DNA samples from 14 strains of H. capsulatum, including the reference strain.

View larger version (64K): [in this window] [in a new window]   FIG. 3.   Comparison of the specificity of a newly prepared PCR primer pair (Hc-T-s and Hc-T-a) to H. capsulatum and other reference fungi and human gene samples. For PCR conditions, see Materials and Methods. Amplification of a 373-bp band can be seen only from the DNA of the H. capsulatum strain.

Based on this study, it appears that a combination of the present primer set for H. capsulatum and the primer set previously described for P. marneffei (7) should permit rapid diagnosis of both these fungal pathogens which are endemic in Thailand and other geographical regions. In addition, these specific molecular techniques should obviate discrimination of these fungi by biological methods, which have been problematic not only in clinical laboratories in Thailand handling AIDS patients but also in similar hospitals throughout the world.

Nucleotide sequence accession number. The DNA sequence of the 705-bp fragment from H. capsulatum NIH 40-0112-13 was submitted to DDBJ (DNA Data Bank of Japan) under accession no. AB011366.